The murine teratocarcinoma system will be employed to study the regulatory mechanisms controlling tumorigenicity, determination, and differentiation. Cell lines have been established of embryonal carcinoma cells, which are the stem cells of teratocarcinomas. These cells are tumorigenic and retain the ability to differentiate into derivatives of each of the three germ layers. Differentiation can be induced in vitro by a variety of agents including retinoic acid, dimethylacetamide, and bromodeoxyuridine. This differentiation is accompanied by a loss of tumorigenicity. One goal of this research is to identify additional agents which induce differentiation of F9 and PCC3 embryonal carcinoma cells and to characterize the phenotype of the resultant differentiated cells. The major goal of the research is to isolate and characterize variants from these cell lines which still immunologically and biochemically resemble embryonal carcinoma cells but fail to differentiate in response to one or more of the inducers. If the regulatory pathway consists of a series of steps leading first to determination and then to differentiation, it should be possible to isolate a series of variants each blocked in a separate regulatory step. The effect of various mutagenic agents on the frequency and class of variants obtained will be determined. Revertants will be isolated and characterized and the effect of various mutagens on the reversion frequency will be determined. Hybridization analyses will be carried out to determine whether the lesion in the variants behaves in a dominant or recessive fashion. The number of complementation groups comprised by the recessive variants will be determined. This number will indicate the complexity of the regulatory pathway. Preliminary experiments will be carried out to determine the biochemical lesion of the variants. Attempts will be made to isolate temperature-sensitive developmental variants. One developmental variant (5C) has been isolated which displays an altered response to retinoic acid. This variant will be analyzed in detail.